CN110426471B - Method for measuring biotin content in infant food - Google Patents
Method for measuring biotin content in infant food Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/04—Preparation or injection of sample to be analysed
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- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a method for determining biotin content in infant food, which is based on ultra-high performance liquid chromatography-isotope dilution mass spectrometry and establishes a method for analyzing and determining biotin content by comparing different extraction and purification methods of biotin in infant formula milk powder and infant cereal auxiliary food. The method has short analysis period and high sensitivity and accuracy, and is suitable for measuring the biotin content in infant formula milk powder and cereal auxiliary food. The invention has important significance for monitoring the biotin strengthening dose and correctly marking the content level of the biotin in production enterprises, and can also provide powerful technical support for government supervision.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a method for determining biotin content in infant food.
Background
Biotin (vitamin) also called vitamin B7Vitamin H, which comprises a carboxyl side chain (containing 5 carbon atoms) and two five-membered heterocycles in the chemical structure, is a bicyclic compound. The side chain carboxyl group binds to the lysine residue epsilon-NH 2 of the zymoprotein in vivo, and functions as a coenzyme. The participation of biotin in the metabolism of proteins, fats and carbohydrates is one of the essential B-group water-soluble vitamins for maintaining the normal functions and metabolic activities of the human body. Biotin may have 8 different isomers, but only D-biotin has biological activity, and the addition form of biotin is also specified in GB 14880-2012 "national standard for food safety standards for the use of dietary supplements".
The literature reports that some biotin is synthesized by human intestinal microorganisms, so that biotin is rarely deficient in adults. However, the intestinal function of infants is weak and the infants cannot synthesize the biotin, and the infants have single dietary types, so the biotin deficiency is easily caused. If the infant lacks biotin, serious neurological symptoms such as mania, sleepiness, growth retardation, glossitis and dermatitis can be caused, so that the infant food needs to be fortified with biotin. The addition amount of biotin in national standards GB 10765-2010, GB 10767-2010 and GB 10769-2010 of food safety is definitely specified, so that the biotin also becomes a necessary item in infant formula milk powder and infant cereal auxiliary food. Therefore, the accurate determination of the biotin content in the infant food provides a powerful technical support for the biotin-intensified dose control of production enterprises and government supervision.
The biotin determination method includes many methods, mainly including microbiological method, enzyme linked immunosorbent assay, spectrophotometry, fluorescence immunochromatography, biosensor method, capillary electrophoresis, liquid chromatography-mass spectrometry, and the like. Among them, the enzyme-linked immunosorbent assay has strong specificity, but the reagent is expensive. The reproducibility of the biosensor method is poor; spectrophotometry has low sensitivity. The reliability of the fluorescence method has a defect. Capillary electrophoresis methods have difficulties in the micro-preparation of samples and the control of electroosmosis. The microbiological method is an analysis method of national standard GB 5009.259-2016, and although the method has high sensitivity, the method has the defects of long detection period, high experimental condition requirement, poor parallelism among samples, difficult strain preservation, poor repeatability, complex operation and the like, and is difficult to widely use. The limitation of high performance liquid chromatography is that biotin has no typical purple, because biotin has no typical ultraviolet and fluorescent chromophores, the sensitivity is low, some samples with low biotin content and large matrix interference cannot be measured, and the qualitative effect of a simple ultraviolet detector or a fluorescent detector is not ideal. The ultra-high performance liquid chromatography-mass spectrometry combined method has the advantages of high sensitivity, good selectivity, high analysis speed, high sensitivity and accuracy and the like, and is more suitable for measuring samples with low content and complex matrix.
The main raw materials of the infant formula milk powder are milk and milk protein products, the infant cereal auxiliary food is one or more cereals (such as wheat, rice, barley, oat, rye, corn and the like) as the main raw materials, the difference between the two matrixes is large, and the content of added biotin is low. The infant formula milk powder contains a large amount of protein, fat and phospholipid, the phospholipid is difficult to remove in the traditional pretreatment process, the matrix interference is large, and the accuracy of an analysis result is influenced. Biotin contained in grains in the infant cereal auxiliary food exists mainly in a form of being combined with protein, the method for measuring biotin in common infant formula milk powder reported in the literature has low extraction efficiency, the biotin content is lower than that of the infant formula milk powder, the influence of matrix is more obvious, and the biotin content is difficult to accurately measure.
Disclosure of Invention
The invention provides a method for measuring biotin content in infant food, which aims to solve the problems of low biotin content, complex matrix and difficult accurate measurement in the infant food.
The invention provides a method for measuring biotin content in infant food, wherein the infant food comprises infant formula milk powder and infant cereal auxiliary food, and the method comprises the following steps:
(1) respectively preparing a biotin standard stock solution and a biotin isotope standard stock solution from a biotin standard substance and a biotin isotope standard substance; and (3) diluting the biotin standard stock solution and the biotin isotope standard stock solution respectively by using a methanol water solution to obtain a biotin standard intermediate solution and a biotin isotope standard intermediate solution.
(2) And respectively taking biotin standard intermediate solutions with different volumes, respectively adding the biotin isotope standard intermediate solutions with the same volume, and fixing the volume by using the initial mobile phase to obtain standard series working solutions.
(3) Weighing a certain mass of infant food sample, adding biotin isotope standard intermediate solution, performing isoelectric point precipitation or high-temperature hydrolysis, and filtering with filter paper to obtain sample extract.
(4) Connecting a solid phase extraction column to a solid phase extraction device, sampling an extract, adding an acetic acid solution, performing vortex mixing, then sampling, rinsing with water and a methanol solution, eluting with acetonitrile-methanol, collecting all eluates, blowing nitrogen to be nearly dry, re-dissolving with an initial mobile phase, and filtering with an organic phase microporous filter membrane to obtain a sample to-be-detected solution.
(5) Respectively sucking the standard series working solution and injecting the standard series working solution into an ultra high performance liquid chromatography-mass spectrometer to obtain a chromatogram of the standard series working solution; and establishing a biotin standard curve according to the ratio of the peak area of the biotin chromatographic peak to the peak area of the biotin isotope chromatographic peak in the chromatogram of the standard series working solution and the biotin concentration.
(6) Absorbing the liquid to be detected of the sample and injecting the liquid into an ultra-high performance liquid chromatography-mass spectrometry combination instrument to obtain a sample chromatogram; and substituting the ratio of the peak area of the biotin chromatographic peak to the peak area of the biotin isotope chromatographic peak in the sample chromatogram into a biotin standard curve to obtain the biotin content in the infant food sample.
Preferably, the chromatographic conditions of the ultra performance liquid chromatography-mass spectrometer are as follows: a chromatographic column: c18 liquid chromatographic column with column length of 100mm, column inner diameter of 2.1mm and filler granularity of 1.7 μm; column temperature: 40 ℃; sample introduction amount: 5 mu L of the solution; mobile phase: 0.1% formic acid solution and acetonitrile; flow rate: 0.3 mL/min.
Preferably, the mass spectrum conditions of the ultra performance liquid chromatography-mass spectrometer are as follows: an ion source: an electrospray ion source; ionization mode: a positive ion mode; capillary voltage: 3.0 kV; taper hole voltage: 30V; the temperature of desolventizing gas is 450 ℃; ion source temperature: 150 ℃; desolventizing air flow rate: 850L/h: taper hole blowback air flow rate: 150L/h; collision airflow rate: 0.12 mL/min; an acquisition mode: and (5) monitoring multiple reactions.
Preferably, in the step (3), when the infant food sample is an infant formula milk powder sample, a certain mass of the infant formula milk powder sample is weighed in a colorimetric tube, after biotin isotope standard intermediate liquid is added, water with the temperature of 45-50 ℃ is added, the infant formula milk powder sample is placed in ultrasonic waves for ultrasonic extraction for about 25min, the ultrasonic waves are cooled to room temperature, the pH value is adjusted to 1.7 +/-0.1 by hydrochloric acid solution, the sample is placed for about 2min, the pH value is adjusted to 4.5 +/-0.1 by sodium hydroxide solution, the volume is fixed by water, and the sample extracting solution is obtained after filtering by filter paper.
Preferably, in the step (3), when the infant food sample is an infant cereal auxiliary food sample, weighing a certain mass of the infant cereal auxiliary food sample in a colorimetric tube, adding a biotin isotope standard intermediate solution, adding 0.1mol/L sulfuric acid solution, sufficiently shaking up, hydrolyzing at 121 ℃ for 30min, cooling to room temperature, adjusting pH to 4.5 +/-0.2 with 1mol/L sodium hydroxide solution, adding water to a constant volume, and filtering with filter paper to obtain a sample extracting solution.
Preferably, in step (4), the solid phase extraction column is a QASIS PRiME HLB solid phase extraction column.
The invention aims at samples of two different matrixes of infant food, including infant formula milk powder and infant cereal auxiliary food, and establishes an analysis method for determining the biotin content in the samples by using a liquid chromatography-tandem mass spectrometry method. The invention has the following beneficial effects:
(1) the detection efficiency is improved. The microbial analysis method adopted by the national standard GB 5009.259-2016 has a long detection period, and only inoculation and culture need to be carried out for 19-20 hours; the operation is complex, the operation is required under the aseptic condition, and the requirement on the experimental environment is severe; the parallelism among samples is not good; difficult preservation of the strain, etc. The method has the advantages of simple pretreatment process and short analysis period, greatly improves the detection efficiency, and is suitable for batch detection of the biotin content in infant formula milk powder and infant cereal auxiliary food.
(2) The solid phase extraction column is adopted for purifying the infant food for the first time, so that the influence of matrix interferents such as fat, salt, phospholipid and the like in a sample on the measurement of biotin is effectively removed, the influence of matrix effect is reduced, and the accuracy of analysis is improved; the pollution to the instrument and the chromatographic column is reduced.
(3) The method has high sensitivity, good accuracy and good precision. Is suitable for measuring the low-content biotin in the infant formula milk powder and the infant cereal auxiliary food.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without any inventive exercise.
FIG. 1 is a graph of the effect of different extraction regimes on biotin extraction;
FIG. 2 is a graph of the effect of sample extract acidity;
FIG. 3 shows the effect of purification and lack of purification of different samples on the matrix effect;
FIG. 4 is a MRM and TIC chromatogram of a standard solution (10ng/mL) provided by the present invention.
Detailed Description
The invention provides a method for measuring biotin content in infant food, which aims to solve the problems of low biotin content, low matrix and difficult accurate measurement in the infant food. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
1 instruments, reagents and materials
ACQUITYTMThe ultra-high performance liquid chromatograph and the Xevo TQ-S mass spectrometer are provided with an electrospray ionization (ESI) source and MasslynxTMChromatography workstations, Waters corporation; ningbo Xinzhi Biotech limited of ultrasonic cleaner; MS3 vortex mixer IKA; N-EVAP-45 nitrogen blowing instrument, Organomation, Inc; SQP-electronic balance sedorischiatics instruments ltd; Milli-Q ultra pure Water System Millipore, USA; model GM-300 pulverizer Leichi, Germany.
D-biotin standard substance, dr. ehrenstorfer, germany; biotin-d 2, deuterated biotin isotope available from ISOREAG; experimental infant food samples (including infant formula and infant cereal adjuncts) were purchased from local supermarkets. Methanol, acetonitrile (chromatographically pure) Fisher corporation, USA; formic acid (chromatographically pure) Sigma-Aldrich, USA; the water is prepared by a Milli-Q ultrapure water machine; nitrogen (> 99.999%); chemical reagents of sulfuric acid and acetic acid (super pure) national drug group; sodium hydroxide (analytical pure) chemical reagents of the national pharmaceutical group, ltd.
ACQUITY UPLC BEH C18 column (100 mm. times.2.1 mm,1.7 μm) manufactured by Waters corporation, USA; QASIS PRIME HLB solid phase extraction column (60mg, 3mL) from Waters corporation, USA; organic microporous filter membrane (0.22 μm) Shanghai' an spectral scientific instruments, Inc.
2 preparation of Standard solution
D-Biotin Standard stock solution (100. mu.g/mL): 5mg (to the nearest 0.1mg) of biotin standard was weighed accurately into a 50mL volumetric flask and the volume was scaled up with a methanol solution (50%). Stored in brown glass bottles, sealed at-20 ℃ for 1 year.
D-Biotin Standard intermediate (100 ng/mL): taking a proper amount of 1.00mL biotin standard stock solution into a 10mL volumetric flask, diluting with a methanol solution (50%) and fixing the volume to a scale; then methanol solution (50%) is gradually diluted to 100ng/mL and stored in a brown glass bottle for 1 month at 2-4 ℃.
Biotin-d 2 Standard stock solution (100. mu.g/mL): 5mg (to the nearest 0.1mg) of biotin-D2 was weighed out accurately into a 50mL volumetric flask and the volume was fixed with a methanol solution (50%). Stored in brown glass bottles, sealed at-20 ℃ for 1 year.
Biotin-d 2 Standard intermediate (2. mu.g/mL): 2.00mL of biotin-d 2 standard stock solution was taken in a 100mL volumetric flask, diluted with methanol solution (50%) and fixed to the volume to the scale, stored in a brown glass bottle and stored at 2-4 ℃ for 1 month.
Preparing a standard series of working solutions: accurately sucking a proper amount of biotin-d 2 standard intermediate solution 75 mu L, D-biotin standard intermediate solution into a 10mL volumetric flask, preparing standard series working solutions with mass concentrations of 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 15ng/mL, 20ng/mL and 25ng/mL by using initial flowing phase, and preparing the working solutions in situ when in use.
3 sample preparation
Uniformly mixing the powdery samples, and directly sampling; and (4) grinding the flaky and granular samples into powder by a sample grinder, and storing the powder in a sample bottle in a dark place. Immediately or stored in a refrigerator at 4 ℃ for 1 week.
4 sample pretreatment
4.1 extraction
4.1.1 extraction of infant formula milk powder
If the infant food sample is the infant formula milk powder sample, weighing 1g (accurate to 0.001g) of the infant formula milk powder sample in a 50mL colorimetric tube, adding 375 mu L of biotin-d 2 standard intermediate solution, adding about 25mL of water with the temperature of 45-50 ℃, placing in ultrasonic waves for ultrasonic extraction for about 25min, cooling to room temperature, adjusting the pH to 1.7 +/-0.1 by using hydrochloric acid solution, placing for about 2min, adjusting the pH to 4.5 +/-0.1 by using sodium hydroxide solution, fixing the volume to 50mL by using water, and filtering by using filter paper to obtain the infant formula milk powder sample extracting solution.
Biotin is a water-soluble vitamin, and both infant formula powders and cereal adjuncts contain proteins, so that protein removal is required at the same time as extraction of the target. The water-soluble vitamins in the infant formula milk powder are mainly subjected to two modes of isoelectric point precipitation and acid hydrolysis to remove proteins. The extraction effect of hydrolysis at 121 ℃ for 30min after isoelectric precipitation and dissolution in 0.1mol/L sulfuric acid was examined (Table 1). Researches show that the protein precipitation effect is good in the two extraction modes, and the filtrate is uniform and transparent and can smoothly pass through the filter membrane. As can be seen from Table 1, there was no significant difference in the effect on recovery and precision; the matrix effect is below 15%, and the influence is small. Since the isoelectric precipitation operation is relatively simple, and the proteins in the infant formula powder mainly comprise whey protein (isoelectric point is pH 1.7) and casein (isoelectric point is pH 4.5), the proteins are precipitated by selectively adjusting pH 1.7 and pH 4.5.
Table 1 influence of different extraction methods on recovery, precision and matrix effect in infant formula (n ═ 6)
4.1.2 extraction of infant formula milk powder
If the infant food sample is the infant cereal auxiliary food sample, weighing 1g (accurate to 0.001g) of the uniformly crushed infant cereal auxiliary food sample into a 50mL colorimetric tube, adding 375 mu L of biotin-d 2 standard intermediate solution, adding 25mL of 0.1mol/L sulfuric acid solution, fully shaking, hydrolyzing at 121 ℃ for 30min, cooling to room temperature, adjusting the pH to 4.5 +/-0.2 by using 1mol/L sodium hydroxide solution, adding water to a constant volume of 50mL, and filtering by using filter paper to obtain the infant cereal auxiliary food sample extracting solution.
As the main raw materials in the formula of the infant cereal auxiliary food are one or more grains (such as rice, millet, wheat, barley, oat, rye, corn and the like), for the products, samples contain protein and starch, and the extraction effect of different enzymolysis, acid hydrolysis and combination modes thereof on biotin in the infant cereal auxiliary food is examined (figure 1). The specific examination conditions are as follows: a. adding water with the temperature of 45-50 ℃ into a sample, dissolving and uniformly mixing, adding 0.1g of amylase and 0.1g of papain, carrying out enzymolysis at the temperature of 55 ℃ for 30min, cooling to room temperature, adjusting the pH of a sample solution to 1.7 +/-0.1, standing for about 1min, and adjusting the pH to 4.5 +/-0.1; b. dissolving a sample by using 0.1mol/L sulfuric acid, hydrolyzing for 30min at 121 ℃, and adjusting the pH of a sample solution to 4.5 +/-0.1; c. after the sample was hydrolyzed in the same manner as b, 1mL of protease-amylase solution (0.2 g of amylase and 0.2g of papain, respectively, and 20mL of water was added and mixed) was added and subjected to enzymolysis at 36. + -. 1 ℃ for 16 hours. As can be seen from figure 1, for the infant cereal auxiliary food, the content (figure 1, A) and recovery rate (figure 1, B) of the biotin in the enzymolysis mode are low, and the enzymolysis results after sulfuric acid hydrolysis and sulfuric acid hydrolysis have no obvious difference. The extraction efficiency, the matrix effect and the inspection period of biotin in the sample are comprehensively considered, and the extraction mode of sulfuric acid hydrolysis is selected in the experiment.
4.2 purification
Connecting a QASIS PRIME HLB solid-phase extraction column to a solid-phase extraction device, taking 1mL of 4.1 sample extracting solution (comprising infant formula milk powder sample extracting solution and infant cereal auxiliary food sample extracting solution) into a 15mL centrifuge tube, adding 1mL of 3% acetic acid solution, performing vortex mixing uniformly, then loading, then respectively leaching with 3mL of water and 3mL of 5% methanol solution, finally eluting with 1mL of acetonitrile-methanol (9:1, V/V), blowing nitrogen at 45 ℃ to be nearly dry, re-dissolving with 1mL of initial mobile phase, and filtering by a 0.22 mu m organic phase microporous filter membrane to obtain a sample to-be-detected solution (comprising the infant formula milk powder sample to-be-detected solution and the infant cereal auxiliary food sample to-be-detected solution).
Infant formula and cereal adjuncts are complex in composition and often contain large amounts of protein, fat and phospholipids. During sample pretreatment, proteins are removed by hydrolysis precipitation, but fats and phospholipids are extracted along with target components. These co-extracts interfere with the measurement of biotin, affect the ionization process of biotin, reduce its ionization efficiency, and thus affect the accuracy of the analysis; and also contaminate the instrument and the column. The structure of biotin comprises two five-membered heterocycles and a carboxyl side chain containing 5 carbon atoms, so that the biotin is suitable for purification of a solid phase extraction column of a reverse phase adsorbent.
Since the biotin chemical structure contains a valeric acid side chain, the aqueous solution has a weak acidic pH value of about 4.5, so when the QASIS PRIME HLB solid phase extraction column is used for purification, the acidity of the sample extracting solution may influence the retention of biotin on the solid phase extraction column. The experiment takes the peak area response value of biotin-d 2 as a reference, and considers the influence of adding 1mL of 3% acetic acid solution to the extract of infant formula milk powder sample and infant cereal auxiliary food sample to adjust acidity and not adding 1mL of 3% acetic acid solution to a solid phase extraction column on biotin retention before loading (figure 2). Experiments show that the response value of the peak area of biotin-d 2 after 1mL of 3% acetic acid solution is added is 4 to 8 times that of the biotin-d 2 without 1mL of 3% acetic acid solution, and the acidity of a sample extracting solution has a large influence on the retention of biotin by a solid phase extraction column. The reason is that after 1mL of 3% acetic acid solution is added, biotin mainly exists in a molecular state, the polarity is weak, and the biotin can be well reserved on a solid-phase extraction column during sample loading; when 1mL of 3% acetic acid solution is not added, biotin mainly exists in an ionic state, the polarity is strong, the biotin is kept weak on a solid-phase extraction column, and most of target substances are lost in the processes of loading and leaching. Therefore, 1mL of 3% acetic acid solution is added before the sample is loaded to increase the adsorption of biotin by the solid phase extraction column.
The infant formula milk powder mainly takes milk and milk protein products as main raw materials, while the infant cereal auxiliary food is mainly prepared from one or more cereals, and the difference of the two matrixes is large; therefore, the experiment takes the matrix effect as a reference, and examines the influence of the purification by using the QASIS PRIME HLB solid phase extraction column and the purification without using the QASIS PRIME HLB on the purification effect of the infant formula milk powder and the infant cereal auxiliary food. As can be seen in FIG. 3, the effect of purging and non-purging on the matrix effect was different for different samples using the QASIS PRIME HLB solid phase extraction column. The effect of the purified and unpurified substrate of the infant formula milk powder is greatly influenced, the effect of the substrate before purification is large, and the effect of the substrate after purification is not obvious; the matrix effect of the infant cereal-based auxiliary food after purification is obviously lower than that before purification, the influence of the matrix effect on the biotin content measurement is effectively reduced, and the accuracy and the sensitivity of an analysis result are improved. The experiment therefore chose to use QASIS PRiME HLB purification.
5 Biotin content analysis
5.1 chromatographic conditions
A chromatographic column: ACQUITY UPLC BEH C18(100 mm. times.2.1 mm,1.7 μm) manufactured by Waters of America; flow rate: 0.3 mL/min; mobile phase: 0.1% formic acid solution (a) and acetonitrile (B); column temperature: 40 ℃; sample introduction amount: 5 μ L. The gradient elution procedure is shown in table 2.
TABLE 2 HPLC gradient elution procedure
The mobile phase composition can influence the separation, peak shape and ionization efficiency of the target compound, and under the same gradient elution condition, the effects of five mobile phases of acetonitrile-0.1% formic acid, acetonitrile-10 mmol/L ammonium formate (containing 0.1% formic acid), acetonitrile-0.1% acetic acid, acetonitrile-10 mmol/L ammonium acetate (containing 0.1% acetic acid) and methanol-0.1% formic acid on the separation, sensitivity and peak shape of the target compound on a BEH C18 chromatographic column are respectively compared. The result shows that when the organic phase is methanol, the biotin peak area response value is low, the number of the impurity peaks is large, and the separation is not good; when the organic phase is acetonitrile, the response values of the peak areas of 0.1 percent formic acid and 10mmol/L ammonium formate (containing 0.1 percent formic acid) biotin are higher than those of 0.1 percent acetic acid and 10mmol/L ammonium acetate (containing 0.1 percent acetic acid), but the addition of the ammonium acetate in the mobile phase does not obviously promote the peak shape and the sensitivity of a chromatographic peak, so that the acetonitrile-0.1 percent formic acid is selected as the mobile phase.
5.2 Mass Spectrometry conditions
An ion source: electrospray ion source (ESI); ionization mode: positive ion mode (ESI +); capillary voltage: 3.0 kV; taper hole voltage: 30V; the temperature of desolventizing gas is 450 ℃; ion source temperature: 150 ℃; desolventizing air flow rate: 850L/h: taper hole blowback air flow rate: 150L/h; collision airflow rate: 0.12 mL/min; an acquisition mode: multiple Reaction Monitoring (MRM); the qualitative ion quantitative ion pair and mass spectrum parameters of biotin are shown in Table 3.
TABLE 3 Mass Spectrometry parameters for Biotin
Note: is a quantitative ion
5.3 detection of Biotin content
Preparing standard series working solutionAnd sequentially injecting the mixture into an ultra-high performance liquid chromatography-mass spectrometry combination instrument according to the concentration from low to high to obtain the chromatograms of the standard series working solutions. And establishing a biotin standard curve according to the ratio of the peak area of the biotin chromatographic peak to the peak area of the biotin isotope chromatographic peak in the chromatogram of the standard working solution and the biotin concentration. And (3) drawing a standard curve by taking the mass concentration (X, ng/mL) of the biotin as a horizontal coordinate and taking the ratio (Y) of the peak area of a biotin chromatographic peak to the peak area of a biotin isotope chromatographic peak, namely the peak area of D-biotin and D2 as a vertical coordinate. The result shows that the linear regression equation is that Y is 1.04308x +0.0133202, R is in the range of 0.5-25 ng/mL2The linear relationship was good at 0.9975. The MRM and TIC chromatograms of the standard solution diagram are shown in figure 4.
After being treated according to the sample pretreatment method, the sample solution to be tested (including the infant formula milk powder sample solution to be tested and the infant cereal auxiliary food sample solution to be tested) is absorbed and injected into an ultra-high performance liquid chromatography-mass spectrometry combined instrument, and the chromatogram of the sample solution to be tested is obtained. And substituting the ratio of the peak area of the biotin chromatographic peak to the peak area of the biotin isotope chromatographic peak in the chromatogram of the sample to be detected into a biotin standard curve to obtain the content of biotin in the sample.
The method was used to analyze commercially available infant formula (10 parts) and infant cereal adjuncts (10 parts) labeled biotin-containing. The result shows that the biotin content is respectively 13.2-38.3 mu g/100g of infant formula milk powder and 2.5-31.9 mu g/100g of infant cereal auxiliary food. The method can be used for detecting the biotin content in infant formula milk powder and cereal auxiliary food.
After the pretreatment is carried out according to the pretreatment method, the signal to noise ratio is tested, and the quantitative limit and the detection limit are respectively determined. The quantitative Limit (LOQ) of the target object is obtained with a signal-to-noise ratio S/N of 10 or more, and the detection Limit (LOD) of the target object is obtained with a signal-to-noise ratio S/N of 3 or more. Biotin has an LOD of 0.75. mu.g/100 g and an LOQ of 2.5. mu.g/100 g. The detection limit and the quantitative limit of the method are both lower than those of the microbial method adopted by the national standard GB 5009.259-2016. Can be used for qualitative and quantitative analysis of biotin in samples with low content.
6 evaluation of matrix Effect
Respectively measuring peak area response values of a pure solvent added with the same-level isotope internal standard and a sample solution to be measured, and calculating a relative ratio of the peak area response values to evaluate a Matrix effect (Matrix effect). Matrix effect factor (MEF,%) (a-B)/ax100, wherein MEF is a Matrix effect factor, a is a peak area response value of an internal standard substance in a pure solvent, and B is a peak area response value of the internal standard substance in a liquid to be measured of a sample. The MEF is 0, the matrix effect is not generated, the larger the absolute value is, the stronger the matrix effect is, and the matrix effect is between 15 percent and 15 percent, and the matrix effect influence is not obvious.
Matrix interference effect is the most common in the application of liquid chromatography-tandem mass spectrometry, and the reason for the matrix effect in the liquid chromatography-tandem mass spectrometry is generally considered to be that non-volatile components in the matrix compete with substances to be detected in the process of ionization of the surfaces of droplets, so that the ionization efficiency at an electrospray interface is influenced. The isotope internal standard and the target have the same chemical property and retention time, and the method is the most effective method for eliminating matrix interference and improving the quantitative accuracy in the liquid chromatography tandem mass spectrometry technology, so that the isotope internal standard is adopted for eliminating the matrix interference in the experiment to realize accurate quantification. Experiments show that the MEF value of biotin in the infant formula milk powder and the cereal auxiliary food is between-15% and 15%, and the influence of matrix effect is not obvious.
7 recovery and precision
The recovery rate test was performed by adding 3 standard solutions of different mass concentrations to the infant formula milk powder and the infant cereal auxiliary food, each level was measured 6 times in parallel, and the standard recovery rate and the relative standard deviation were calculated, and the results are shown in table 4. The recovery rate of the method is 96.7-101.4%, the relative standard deviation is 2.29-5.40%, and the method has high recovery rate and good reproducibility. The NIST SRM 1849a sample of the milk powder quality control sample is detected, and the detection result is 1.90mg/kg and is within the deviation range of a calibration value (1.99 +/-0.13 mg/kg).
Table 4 recovery and precision of the procedure (n ═ 6)
In conclusion, the invention is based on the ultra-high performance liquid chromatography-isotope dilution mass spectrometry, and establishes an analysis and determination method for biotin content by comparing different extraction and purification methods of biotin in infant formula milk powder and infant cereal auxiliary food. After biotin in a sample is extracted and purified, gradient elution is carried out on a C18 chromatographic column, electrospray positive ion mode ionization and multi-reaction detection mode are carried out for detection and analysis, and internal standard method quantification is carried out. The result shows that the biotin has good linear relation in the range of 0.5-25 ng/mL and the correlation coefficient (R)2) Is 0.9975; the recovery rate of the added standard is 96.7-101.4%, and the relative standard deviation is 2.29-5.40%. The detection limit (S/N is more than or equal to 3) of the method is 0.75 mug/100 g, and the quantification limit (S/N is more than or equal to 10) is 2.5 mug/100 g. The method has short analysis period and high sensitivity and accuracy, and is suitable for measuring the biotin content in infant formula milk powder and cereal auxiliary food. The invention has important significance for monitoring the biotin strengthening dose and correctly marking the content level of the biotin in production enterprises, and can also provide powerful technical support for government supervision.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure herein. This invention is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (5)
1. A method for determining the biotin content in infant food, which comprises infant formula milk powder and infant cereal auxiliary food, and comprises the following steps:
(1) respectively preparing a biotin standard stock solution and a biotin isotope standard stock solution from a biotin standard substance and a biotin isotope standard substance; respectively diluting the biotin standard stock solution and the biotin isotope standard stock solution by using a methanol aqueous solution to obtain a biotin standard intermediate solution and a biotin isotope standard intermediate solution;
(2) respectively taking biotin standard intermediate solutions with different volumes, respectively adding the biotin isotope standard intermediate solutions with the same volume, and fixing the volume by using an initial mobile phase to obtain standard series working solutions;
(3) weighing a certain mass of infant food sample, adding a biotin isotope standard intermediate solution, performing isoelectric point precipitation or high-temperature hydrolysis, and filtering by filter paper to obtain a sample extracting solution;
(4) connecting a solid phase extraction column to a solid phase extraction device, sampling an extracting solution, adding a 3% acetic acid solution, performing vortex mixing, then sampling, leaching with water and a 5% methanol solution, eluting with acetonitrile-methanol, collecting all eluents, blowing nitrogen to be nearly dry, re-dissolving with an initial mobile phase, and filtering with an organic phase microporous filter membrane to obtain a sample solution to be detected;
(5) respectively sucking the standard series working solution and injecting the standard series working solution into an ultra high performance liquid chromatography-mass spectrometer to obtain a chromatogram of the standard series working solution; establishing a biotin standard curve according to the ratio of the peak area of a biotin chromatographic peak to the peak area of a biotin isotope chromatographic peak in the chromatogram of the standard series working solution and the biotin concentration;
(6) absorbing the liquid to be detected of the sample and injecting the liquid into an ultra-high performance liquid chromatography-mass spectrometry combination instrument to obtain a sample chromatogram; substituting the ratio of the peak area of the biotin chromatographic peak to the peak area of the biotin isotope chromatographic peak in the sample chromatogram into a biotin standard curve to obtain the biotin content in the infant food sample;
in the step (4), the solid phase extraction column is a QASIS PRIME HLB solid phase extraction column.
2. The method for determining the biotin content in infant food according to claim 1, wherein the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometer are as follows:
a chromatographic column: c18 liquid chromatographic column with column length of 100mm, column inner diameter of 2.1mm and filler granularity of 1.7 μm;
column temperature: 40 ℃;
sample introduction amount: 5 mu L of the solution;
mobile phase: 0.1% formic acid solution and acetonitrile;
flow rate: 0.3 mL/min.
3. The method for determining the biotin content in infant food according to claim 1, wherein the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometer are as follows:
an ion source: an electrospray ion source; ionization mode: a positive ion mode; capillary voltage: 3.0 kV; taper hole voltage: 30V; the temperature of desolventizing gas is 450 ℃; ion source temperature: 150 ℃; desolventizing air flow rate: 850L/h: taper hole blowback air flow rate: 150L/h; collision airflow rate: 0.12 mL/min; an acquisition mode: and (5) monitoring multiple reactions.
4. The method according to claim 1, wherein in the step (3), when the infant food sample is an infant formula milk powder sample, a certain mass of the infant formula milk powder sample is weighed in a colorimetric tube, after the biotin isotope standard intermediate solution is added, water with a temperature of 45-50 ℃ is added, the sample is placed in ultrasonic waves for ultrasonic extraction for about 25min, after the sample is cooled to room temperature, the pH value is adjusted to 1.7 ± 0.1 by hydrochloric acid solution, after the sample is placed for about 2min, the pH value is adjusted to 4.5 ± 0.1 by sodium hydroxide solution, the volume is determined by water, and after the sample is filtered by filter paper, the sample extract is obtained.
5. The method for determining biotin content in infant food according to claim 1, wherein in step (3), when the infant food sample is an infant cereal-based food sample, a certain mass of the infant cereal-based food sample is weighed in a colorimetric tube, after the biotin isotope standard intermediate solution is added, 0.1mol/L sulfuric acid solution is added and shaken well, and is hydrolyzed at 121 ℃ for 30min, and is cooled to room temperature, and after the pH is adjusted to 4.5 ± 0.2 with 1mol/L sodium hydroxide solution, the volume is fixed with water, and after being filtered by filter paper, a sample extract is obtained.
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